Detailed procedure and tips for UV cross-linking and immunoprecipitation (CLIP).

Interest in RNA-protein interactions is booming as we begin to appreciate the role of RNA, not just in well-established processes such as transcription, splicing, and translation, but also in newer fields such as RNA interference and gene regulation by non-coding RNAs.

CLIP is an antibody-based technique used to study RNA-protein interactions related to RNA immunoprecipitation (RIP), but differs from RIP in the use of UV radiation to cross-link RNA binding proteins to the RNA that they are bound to.

This covalent bond is irreversible, allowing stringent purification conditions. Unlike RIP, CLIP provides information about the actual protein binding site on the RNA.

iCLIP protocol:

Remove media and add ice-cold PBS to cells (e.g. use cells grown in a 10 cm plate for three experiments and add 6 ml PBS).

Remove lid, place on ice and irradiate once with 150 mJ/cm2 at 254 nm using a stratalinker.

*One or more negative controls should be maintained throughout the complete experiment. Knockout cells or tissue as well as non-cross-linked cells are good negative controls, while knockdown cells are not recommended.

Harvest cells with a cell scraper and transfer cell suspension to microtubes (e.g. 2 ml to each of three microtubes).

Pellet cells (spin at top speed for 10 sec at 4°C), then remove supernatant.

Snap-freeze cell pellets on dry ice and store at -80°C until use.

*Experiment could take up to a week. Avoid multiple cycles of freeze thaw.

Spin at 4°C at 22,000 g for 20 min and carefully collect the cleared supernatant (leave about 50 μl lysate with the pellet).

*Each member of the laboratory should use their own set of buffers and reagents to easier identify potential sources of contamination. Ideal conditions for the RNase digestions may need to be optimized for every new batch of RNase.

Immediately place on a magnet to precipitate the empty beads and load the supernatant on the gel.

6. SDS-PAGE and membrane transfer

Load samples as well as a pre-stained protein size marker (5 μl) on a precast 4-12% Bis-Tris gel, and run the gel for 50 min at 180 V in 1x MOPS running buffer (according to manufacturer's instructions).

Transfer the protein-RNA complexes from the gel to a nitrocellulose membrane using a wet transfer apparatus (transfer 1 h at 30 V depending on manufacturer's instructions).

*More information on SDS-PAGE and transfer can be found under our Western blot protocols.

Following the transfer, rinse the membrane in PBS buffer, then wrap it in cling film and expose it to a film at -80°C for 30 min, 1 hr and then overnight.

*A fluorescent sticker next to the membrane later allows to align the film and the membrane. The success of the experiment can be monitored at the autoradiograph of the protein-RNA complex after membrane transfer

Figure 1: Image of a typical autoradiograph

*Control experiments should give no signal on autoradiograph. In the autoradiograph of the low-RNase samples, diffuse radioactivity should be seen above the molecular weight of the protein. For high-RNase samples, this radioactivity is focused closer to the molecular weight of the protein.

7. RNA isolation

Isolate the protein-RNA complexes from the membrane using the autoradiograph from step 6.4 as a mask. Cut this piece of membrane into several small slices and place them into a 1.5 ml microtube.

Run the gel for 40 min at 180 V depending on manufacturer’s instructions. This leads to a reproducible migration pattern of cDNAs and dyes (light and dark blue) in the gel (see figure 2).

Use a razor blade to cut (red line) three bands of cDNA fractions at 120-200 nt (high [H]), 85-120 nt (medium [M]) and 70-85 nt (low [L]). Start by cutting in the middle of the light blue dye, divide the medium and low fractions and trim the high and low fractions. Use vertical cuts guided by the pockets and the dye to separate the different lanes (in figure 2, 1-4). The marker lane (m) can be stained and imaged to control sizes after the cutting. Fragment sizes are indicated on the right.

Add TE (400 μl) and crush the gel slice into small pieces using a 1 ml syringe plunger. Incubate shaking at 1,100 rpm for 2 hr at 37°C.

Place two 1 cm glass pre-filters into a Costar SpinX column and transfer the liquid portion of the sample to the column. Spin for 1 min at 13,000 rpm into a 1.5 ml tube.

*Avoid contamination with PCR products from previous experiments by spatially separating pre- and post-PCR steps. Ideally, analysis of PCR products and all subsequent steps should be performed in a separate room.

11. PCR amplification

Spin down and wash the samples (see 8.1), then resuspend the pellet in water (19 μl).

*The primer sequences used are for solexa sequencing, other systems may require adjustment of the primers.

Mix PCR product (8 μl) with 5x TBE loading buffer (2 μl) and load on a precast 6% TBE gel. Stain the gel with SYBR Green I and analyze the PCR products with a gel imager; this allows monitoring of the success of the experiment prior to sequencing of the iCLIP library.

The gel image of the PCR products should show a size range that corresponds to the cDNA fraction (high, medium or low) purified in step 9.4.

*Note that the PCR primers P3Solexa and P5Solexa introduce an additional 76 nt to the size of the cDNA. Primer dimer product can appear at about 140 nt.

The barcode in the Rclip primers allow to multiplex different samples before submitting for high throughput solexa sequencing.

Submit 15 μl of the library for sequencing and store the rest.

*Control experiments should give no products after PCR amplification, and high-throughput sequencing of control libraries should return very few unique sequences